Surgical aids containing pharmaceutically acceptable fixed-dried human blood platelets

ABSTRACT

Fixed-dried human blood platelets and processes for preparing them are disclosed. The platelets, upon reconstitution: adhere to thrombogenic surfaces; do not adhere to non-thrombogenic surfaces; undergo shape change (spreading) upon adhering to a thrombogenic surface; adhere to one another to form a hemostatic plug upon adhering to a thrombogenic surface; and release their granular contents. Surgical aids containing such platelets are also disclosed. The platelets are preferably fixed by a fixative such as formaldehyde, paraformaldehyde, or glutaraldehyde, or fixed by a permanganate fixate. The platelets are preferably dried by lyophilization.

This invention was made with government support under Grant No. 5-42104awarded by the Office of Naval Research. The government has certainrights in the invention.

This application is a divisional of application Ser. No. 08/325,424,filed Nov. 28, 1994, now U.S. Pat. No. 5,651,966, which is aContinuation-in-Part application of Ser. No. 07/891,277, filed May 29,1992, now abandoned, the disclosure of which is incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to fixed-dried blood platelets suitablefor administration to human patients and methods of treating woundtissue by the topical application of fixed-dried platelets.

BACKGROUND OF THE INVENTION

The use of platelet concentrates in transfusion medicine has become wellestablished during the past thirty years. However, the rapid loss ofplatelet function during the storage period and risk of bacterialcontamination has greatly complicated management of an effectiveinventory of platelet concentrates in blood banks. In many settings, thelimited shelf life of platelet concentrates has drastically reducedtheir usage.

E. Klein et al., J. Pediatrics 49, 517-522 (1956), describe thepreparation and administration of lyophilized platelet material tochildren with acute leukemia and aplastic anemia. Pain and venospasm atthe site of infusion were noted. The limited effectiveness of thesematerials is shown in Table 2 therein. After more than thirty years,these materials have not led to a useful therapeutic treatment.

In order to make platelet transfusion therapy more manageable for bloodbanks, there has been considerable interest in devising means fordiminishing or delaying the loss of platelet function during the storageperiod. One approach has been in the context of the development ofplasma-free storage media. See, e.g., S. Holme, U.S. Pat. No. 4,695,460.Another approach has been to employ biochemical techniques to stabilizethe platelets. See, e.g., A. Bode et al., U.S. Pat. No. 4,994,367. Whilethese techniques provide useful extension of shelf life, they do notprovide a shelf life extended for prolonged periods of time. Finally,the preparation of platelet membrane microvesicles from, among otherthings, outdated platelets is described in F. Chao, U.S. Pat. No.5,185,160.

Fixed-dried blood platelets for use in diagnostic assays are disclosedin U.S. Pat. No. 4,287,087 to Brinkhous et al. While such fixed-driedplatelet preparations can be stored for prolonged periods of time fordiagnostic purposes, they have not heretofore been provided in a formfor human pharmaceutical use. Accordingly, there is a continuing needfor new means of preparing blood platelet preparations having prolongedshelf lives which are suitable for administration to human patients.

SUMMARY OF THE INVENTION

A first aspect of the present invention is fixed-dried human bloodplatelets which, upon reconstitution: (a) adhere to thrombogenicsurfaces; (b) do not adhere to non-thrombogenic surfaces; (c) undergoshape change (spreading) upon adhering to a thrombogenic surface; (d)adhere to one another to form a hemostatic plug upon adhering to athrombogenic surface; and (e) release their granular contents, such asafter stimulation and/or spreading (e.g., after receiving aphysiological stimulation which would ordinarily cause a metabolicallyactive, live or fresh platelet to release its granular contents, such ascontacting wounded tissue).

A second aspect of the present invention is a pharmaceutical formulationcomprised of a fixed-dried blood platelets preparation. The fixed-driedblood platelet preparation comprises fixed-dried human blood plateletshaving the characteristics set forth above.

A third aspect of the present invention is a method of fixing bloodplatelets to produce fixed-dried blood platelets having thecharacteristics set forth above, and the platelets so produced. Themethod comprises contacting the platelets to a fixative such asformaldehyde, paraformaldehyde, glutaraldehyde, or permanganate (e.g.,by mixing the platelets with a solution thereof) for a time sufficientto fix or stabilize the platelets but insufficient to cause loss of thecharacteristics enumerated above. The platelets are then dried to yieldfixed-dried blood platelets having the characteristics set forth above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows thrombin generation by platelets of the present inventionas compared to fixed-dried platelets of the prior art and unactivatedcontrol platelets.

DETAILED DESCRIPTION OF THE INVENTION

Fixed-dried blood platelets of the present invention may be fixed with acompound selected from the group consisting of formaldehyde,paraformaldehyde and glutaraldehyde. Fixation with such agents requirescareful modification of the procedure set forth in U.S. Pat. No.4,287,087 to avoid loss of viability of the platelets. In general,washed platelets are fixed by incubating them, typically at roomtemperature, for up to 60 minutes in a solution of up to 1.8%paraformaldehyde. As discussed in greater detail below, care must alsobe taken to sufficiently fix the platelets or undue lysis will occurduring drying thereof.

An alternative technique is to fix platelets by incubating the plateletsin a permanganate solution (e.g., sodium permanganate, potassiumpermanganate). In general, washed platelets may be prepared by thistechnique by incubating them for from 5 to 20 minutes in from 0.001 to 1g/dL of KMnO₄ or NaMnO₄ solution, more preferably by incubating them forfrom 5 to 15 minutes in from 0.005 to 0.5 g/dL of KMnO₄ or NaMnO₄solution, and most preferably by incubating them for 8 to 12 minutes infrom 0.005 to 0.05 g/dL of KMnO₄ or NaMnO₄ solution.

Blood platelet preparations for use in preparing pharmaceuticalformulations should be essentially free of extraneous matter,particularly lysed blood platelets which would present free thrombogenicagents to a patient administered the preparation. Hence, care must betaken to sufficiently fix the platelets (without destroying theviability thereof, as indicated by the characteristics set forth above)prior to drying, as undue lysis will otherwise occur during the dryingstep. For example, platelet preparations suitable for use in preparinghuman pharmaceutical formulations preferably show, on reconstitution of10⁹ platelets in one milliliter of solution, less than 10×10⁶microparticles (the fragmentary remains of lysed platelets) permilliliter, and preferably show less than 150 International Units (IU)per liter of lactate dehydrogenase in the supernatant after resuspensionand pelleting (where 2200 IU per liter represents total lysis of 10⁹cells in 1 milliliter).

Drying of platelets after fixation may be carried out by any suitablemeans, but is preferably carried out by lyophilization. Care must betaken to stabilize the platelet preparation prior to drying as anunacceptable level of platelet lysis may otherwise occur. Stabilizationmay be carried out by suspending the platelets in a solution containinga suitable water replacing molecule (or "stabilizers"), such as albuminor trehalose, and then drying the solution. In one embodiment, from 0.1to 20 percent by weight albumin is employed, more preferably from 1 to10 percent by weight albumin is employed, and most preferably from 5 to10 percent by weight albumin is employed. For administration to asubject, the albumin in the preparation should be of the same species asthe subject (e.g., human albumin). In the alternative, the preparationmay be dried with albumin of a different species, the albumin separatedfrom the platelets on reconstitution, and albumin of the same speciesadded back to the reconstituted preparation for administration to thesubject, but care should be taken to remove all non-species specificalbumin as it may be antigenic in the subject being treated.

Pharmaceutical formulations of the present invention may simply comprisedried (preferably lyophilized) platelets, pyrogen-free and sterile in asterile aseptic package. Albumin may be included, as noted above.Pharmaceutical formulations may also comprise a platelet preparation ofthe present invention reconstituted in a pharmaceutically acceptablecarrier. Any aqueous carrier which rehydrates the platelets so that theypossess the characteristics enumerated above and are suitable forintravenous injection may be used (e.g., sterile, pyrogen free,physiological saline solution). Additional agents, such as buffers,preservatives, and other therapeutically active agents, may also beincluded in the reconstituted formulation. See, e.g., U.S. Pat. No.4,994,367 (the disclosure of which is incorporated herein by reference).

Reconstituted pharmaceutical formulations of the present invention aretypically administered to human patients by intravenous injection.Patients in need of such treatment include patients afflicted withthrombocytopenia (including washout thrombocytopenia), patientsafflicted with hemorrhagic platelet dysfunction, and trauma victimsexperiencing severe bleeding. The amount of the pharmaceuticalformulation administered will vary depending upon the weight andcondition of the patient, but will typically range from 20 to 350milliliters in volume, and from 1×10⁹ to 3×10⁹ platelets per milliliter(and more preferably from 2×10⁹ to 3×10⁹ platelets per milliliter) inconcentration. Pharmaceutical formulations may be packaged in a sterile,pyrogen free container to provide these volumes and dosages as a unitdose.

Also disclosed herein is a method of enhancing wound healing in asubject in need of such treatment. The method comprises topicallyapplying fixed, dried blood platelets to the wound in an amounteffective to enhance wound healing, wherein said platelets expressplatelet-derived growth factor on the surface thereof. The subject maybe a human subject or an animal subject in veterinary medicine (i.e.,dog, cat, horse, cow, etc.). The platelets may be of any suitablespecies (i.e., human, cow, pig, etc.) but are preferably of the samespecies of origin as the subject undergoing treatment. The platelets maybe prepared by any suitable means so long as they express PDGF, but arepreferably prepared by means such as the methods described herein spthat they release platelet-derived growth factor after stimulationand/or spreading (e.g., after receiving a physiological stimulationwhich would ordinarily cause a metaabolically active, live or freshplatelet to release its granular contents, such as contacting woundedtissue). Any type of wound may be treated by the method, includingabrasions, incisions, punctures, lacerations, burns, etc. The wound maybe a wound to skin tissue, or may be a wound,to the tissue of anotherorgan, such as incisions in internal organs such as intestine, spleen,liver, etc., as encountered during surgery. The platelets may be appliedto the wound by any suitable means, such as by sprinkling or sprayingthe platelets onto the wound, or may be applied by means of a surgicalaid as discussed below. Where the platelets are sprinkled directly ontothe wound, the wound may optionally then be sprayed with a clearpolymeric adhesive material, or then covered with any other suitablebandage or dressing. The dosage of platelets should be at least 0.5 to1×10⁹ platelets per one square-centimeter of surgical aid surface areaor wound surface area. The upper limit of dosage is not particularlycritical, but will generally be 5 to 10×10⁹ platelets per one squarecentimeter of surgical aid surface area or wound surface area.

Fixed, dried blood platelets may be applied to a wound by means of asurgical aid, such as a wound dressing or bandage, a suture, a fabric, aprosthetic device, etc. All such aids comprise, in combination, a solid,physiologically acceptable substrate material, and fixed, dried bloodplatelets carried by (e.g., applied as a coating to or impregnated in)the substrate material, wherein the platelets express platelet-derivedgrowth factor on the surface thereof. Typically, such surgical aids areprovided in sterile form packaged in a sterile container. The surgicalaid substrate may be coated with the platelets prior to packaging (i.e.,by sprinkling dried platelets onto the substrate or drying (e.g.,lyophilizing) the substrate in an aqueous preparation of the fixedplatelets or with an aqueous preparation of the fixed platelets carriedthereon so that some of the platelets adhere to the substrate), packagedwith the platelets so that some of the platelets adhere to the substratein the package, adhered to the substrate with a suitable adhesivematerial, or simply sprinkled onto the surgical aid prior to applicationthereof to the subject.

The surgical aid substrate may take any form or be of any solidmaterial, hydrophobic or hydrophilic, which is physiologicallyacceptable. Sutures, for example, can be monofilament or braided, can bebiodegradable or nonbiodegradable, and can be made of materials such asnylon, silk, polyester, cotton, catgut, homopolymers and copolymers ofglycolide and lactide, etc. Prosthetic devices, for example, includewoven or extruded tubular structures having use in the repair ofarteries, veins, ducts, asophagi; woven or knitted fabrics which aredrapable or conformable and are useful surgically in hernia repair andin supporting damaged liver, kidney, and other internal organs; pins,screws, and reinforcing plates; heart valves (e.g., fixed pig heartvalves), artificial tendons or cartilage material, etc. Polymericmaterials as described in connection with sutures above canalternatively be cast as a thin film, sterilized, and packaged for useas a wound dressing. Bandages may be made of any suitable substratematerial, such as woven or nonwoven cotton or other fabric suitable forapplication to or over a wound, may optionally include a backingmaterial, and may optionally include one or more adhesive regions on theface surface thereof for securing the bandage over the wound.

The present invention is explained in greater detail in the followingExamples. These Examples are for illustrative purposes only, and are notto be taken as limiting of the invention.

EXAMPLE 1 Preparation of Lyophilized Human Platelets (Protocol 1)

Human platelets are prepared from blood drawn into acid citrate dextrose(ACD) anticoagulant (0.085M trisodium citrate, 0.0702M citric acid,0.111M dextrose, pH 4.5), one part anticoagulant to 5.66 parts blood.Platelets were isolated by differential centrifugation and washed threetimes with acid citrate saline (0.00544M trisodium citrate, 0.154M NaCl,adjusted to pH 6.5 with 0.1 N HCl).

After washing, platelets are fixed by incubating the washed plateletsfrom 100 ml of blood in 5.0 ml of 1.8% paraformaldehyde solution(prepared as 9.0 ml 4% paraformaldehyde solution plus 1.0 ml ACD plus10.0 ml 0.135M NaH₂ PO₄) for 45 minutes at room temperature (thefixation time may be extended to 60 minutes). An alternative is toincubate the washed platelets from 100 20 ml of blood in a 1.0%paraformaldehyde solution for 45 minutes at room temperature (thefixation time may be extended to 60 minutes).

To remove the paraformaldehyde, after paraformaldehyde incubation, anequal volume of imidazole buffered saline (0.084M imidazole; 0.146MNaCl, adjusted to pH 6.8 with 1.0 N HCl) , is added to each tube and theplatelets pelleted by centrifugation at 1500 times g for 8 minutes atroom temperature. The supernatant is decanted and the platelets washedby resuspending the platelet pellets in 5-10 ml imidazole bufferedsaline pH 7.35. The wash is repeated twice more to remove theparaformaldehyde. Following the third wash the platelets are resuspendedin a 5% solution of serum albumin (5 gm albumin per 100 ml of citratesaline solution, 00.0054M sodium citrate, 0.154M NaCl, pH 6.5). Theplatelets are counted using a phase contrast microscope and an AmericanOptical Bright-Line Hemocytometer. The platelet concentration isadjusted to 800,000 per cubic millimeter (cam).

Aliquots (10 ml) of concentration-adjusted platelets in the serumalbumin solution are placed in 20 ml glass vials and frozen at -70° C.The platelets are then lyophilized for 12 hours or until a cracked,white powder is evident. The platelet product can also be shell frozenin large quantities of 100 to 500 ml and lyophilized at -40° C. for fourhours, after which the temperature is raised to -25° C. for the durationof the drying time. The lyophilized product is stored at -20° C. to -70°C. until use.

Lyophilized platelets are rehydrated with 0.084M imidazole buffer (nosalt added), adjusted to a pH of 7.35 with 1.0M NaOH. After addition ofimidazole buffer, the solution is allowed to sit, undisturbed forseveral minutes, then gently mixed by rolling or rotating the vial toproduce an even suspension of rehydrated single platelets.

EXAMPLE 2 Preparation of Lyophilized Human Platelets (Protocol 2)

Whole blood is obtained from healthy volunteer donors into commercialblood collection packs (Fenwal 4R6402, Baxter Health Care) containingits standard complement of anticoagulant (CPDA-1). The final volume ofeach unit of citrated whole blood collected is 500 cc.

Each bag of whole blood is centrifuged to obtain platelet-rich plasma(PRP), which is aspirated from the bag and washed by threecentrifugation/resuspension steps in phosphate-buffered saline solution(same as described in Example 1 above). The washed platelets are thencentrifuged again and the pellet treated with a buffered solutioncontaining 1.8% paraformaldehyde (same as described in Example 1 above)for from 45 minutes to 1 hour at room temperature. The yield ofplatelets after removal of the stabilization reagent and furtherplatelet washing to remove paraformaldehyde is 60-80% of the count inthe platelet suspension prior to stabilization. When albumin is notincluded in the washing buffer after stabilization, then the plateletyield falls.

The composition of the final platelet resuspension before freeze-dryingis important to obtaining appropriate yields. In general, an effectiveamount of a stabilizer such as albumin or trehalose in buffered salineis necessary to obtain yields of 85-100% of the platelets through thelyophilization/rehydration steps. Albumin should be included in anamount ranging from 0.1 to 50 g/dL, more preferably an amount rangingfrom 1 to 25 g/dL, and most preferably in an amount ranging from 5-10g/dL. Trehalose should be included in an amount ranging from 0.1-10. M,more preferably from 0.2 to 5M, and most preferably from 0.5-1.0M.Several types of rehydration solutions have been employed withoutnoticeable differences in parameter outcomes: phosphate-buffered salinepH=7.3, tris-buffered saline pH=7.4, imidazole-buffered saline, orUNISOL™ physiologic balanced salt solution.

Typical data for rehydrated platelet preparations prepared as describedin this Example are given in Table 1 below.

                  TABLE 1    ______________________________________    Performance of Rehydrated Platelets in vitro.    Aggregation         Percent Platelets Remaining    Studies             Unaggregated    ______________________________________    1.5 mg/mL ristocetin                        12-15% (strong response)    10 μM ADP        42-85% (weak response)    8 μg/mL collagen 25-60% (medium response)    Flow Cytometry Studies                        Percent Platelets with Normal                        Fluorescence    GPIb (AN-51, SZ-2, SZ-1, MoAbs                        90-97% (Equivalent to fresh                        platelets)    GPIIbIIIa (10E5 MoAb)                        98-99% (Equivalent to fresh                        platelets)    ______________________________________

The microparticle count after rehydration of platelet preparationsprepared as described herein was from 4.5 to 5.0×10⁶ /mL. The hypotonicshock test response for platelets prepared as described in this Examplewas 0.030-0.0036 OD/min (0.100-0.150 for fresh platelets) . Releasedlactate dehydrogenase (LDH), the amount of LDH in the supernatant afterresuspension of 10⁹ platelets in 1 ml of solution, was from 50 to 200IU/L (>150 or 250 indicates significant cytoplasmic leakage).

EXAMPLE 3 Preparation of Lyophilized Human Platelets With PermanganateStabilization

Whole blood is obtained from healthy donors into commercial bloodcollection packs (Fenwal 4R6402, Baxter HealthCare) containing thestandard complement of anticoagulant (CPDA-1). The final volume of eachunit of citrated whole blood collected is 500 cc. Each bag of wholeblood is centrifuged to obtain platelet rich plasma, (PRP) which wasaspirated from the bag and washed by three centrifugation/resuspensionsteps in a phosphate-buffered saline solution as given in Example 1above. The washed platelets were resuspended in one-tenth volumebuffered saline and added dropwise to permanganate solution comprising aphosphate-buffered saline solution containing KMnO₄ or NaMno₄ at a finalconcentration of 0.01 g/dL. The suspension of platelets were incubatedin the permanganate solution for 10 minutes at room temperature, andthen washed twice as above with 0.1-5.0 g/dL albumin in the buffer toremove the permanganate. The loss of platelets during permanganatetreatment and subsequent washing was only 10 to 20%.

To maintain yields of 70-100% after lyophilization/rehydration, it isnecessary to include in the final resuspension solution prior tolyophilization a stabilizer such as trehalose or albumin, preferably inthe ranges given above. Rehydration solution composition is not criticalbut should be isotonic and buffered to pH 7.3-7.4 (same as forparaformaldehyde-fixed platelets). Typical data upon analysis ofrehydrated permanganate-fixed platelets are presented in Table 2 below.

                  TABLE 2    ______________________________________    Performance of Rehydrated Platelets in vitro.    Aggregation         Percent Platelets Remaining    Studies             Unaggregated    ______________________________________    1.5 mg/mL ristocetin                        15-32% (strong response)    10 μM ADP        27-42% (medium response)    8 μg/mL collagen 27-50% (medium response)    Flow Cytometry Studies                        Percent Platelets with Normal                        Fluorescence    GPIb (AN-51, SZ-2, SZ-1,                        91-99% (Equivalent to fresh    MoAbs)              platelets)    GPIIbIIIa (10E5 MoAb)                        95-99% (Equivalent to fresh                        platelets)    ______________________________________

Microparticle count after rehydration of platelet preparations fixed bythe permanganate process described in this example was 2.6-4.0×10⁶ /mL.The hypotonic shock test response for platelets prepared by this processwas 0-0.030 OD/min (0.100-0.150 for fresh platelets). Released LDH was50-200 IU/L.

EXAMPLE 4 COMPARATIVE EXAMPLE A Use of Activation Markers toCharacterize Paraformaldehyde Stabilized Platelets

The purpose of this Example is to demonstrate that platelets fixed withparaformaldehyde in accordance with the present invention release theirgranular contents after contacting a thrombogenic surface, while priorart platelets do not. The platelet preparations examined were stabilizedwith 1.0% paraformaldehyde for 60 minutes (paraform. 1) and with 1.8%paraformaldehyde for 60 minutes (paraform. 2); these were compared toplatelets prepared as described in the Brinkhous et al. patent, i.e.,fixed with 2% paraformaldehyde for 120 minutes (Brinkhous).

The markers employed in the tests described in Table 3, CD62 and GP53,are commercially available antibodies purchased from Becton-Dickinson,Inc. and are used to indicate the presence on the surface of theplatelet of antigens released from platelet granules. The presence ofgranule-released antigens on a platelet surface is taken as evidence ofplatelet activation. Antibodies against these antigens are referred toas activation markers. Antibodies against Platelet-derived growth factor(PDGF) are used to detect platelet membrane bound PDGF. The PDGFantibody was purchased from Genzyme (Cambridge, Mass.). The vessels usedin the Baumgartner adhesiveness tests were obtained from a normal dog.

Experiments were carried out to demonstrate the activatability ofseparate lyophilized platelet preparations by incorporating them intofresh whole blood made free of native platelets by differentialcentrifugation for comparison to fresh, unsubstituted whole blood foruse in annular perfusion chambers. Blood was collected into citrateanticoagulant (CPDA-1) from normal human donors. Two 1 cm strips ofcanine arterial vessel were everted on a tapered rod and inserted into arecirculation loop driven by a peristaltic pump at 130 mL/minute. Theloop was perfused first with buffer, then the blood (with either freshor lyophilized platelets) for 5 minutes at room temperature, followed bya 2 minute perfusion with 2% paraformaldehyde to fix adherent plateletsto the vessel permanently. Platelets on the vessel surface were detectedby additional of a fluorescent monoclonal antibody to GPIIbIIIa.Adherence was quantified by epi-fluorescence microscopy as an estimateof the percent of vessel surface covered by fluorescent cells. Also, theremaining blood (with non-adherent platelets) was sampled to makeplatelet-rich plasma (PRP). The PRP was further fixed with 2%paraformaldehyde for 1-2 hours at room temperature before incubationwith florescence-labelled monoclonal antibodies to CD62 or GP53 or PDGF.The presence of these markers on the surface of platelets in the samplewas detected by standard flow cytometry on a Becton Dickinson FACSCAN™flow cytometer. Quantitation of results was expressed as the percentageof platelets with fluorescence greater than the background recorded witha non-specific control antibody (non-immune mouse IgG2₈). The findingswere compared on blood samples taken just prior to or just afterinitiation of perfusion of the vessel strips. The results of thiscomparison are shown in Table 3.

                  TABLE 3    ______________________________________    Comparison of Paraformaldehyde Stabilized Platelets    with Activation Markers.              Percent Positive Platelets    Marker      Pre-Circulation                           Post-Circulation    ______________________________________    1.0% Paraformaldehyde/60 minutes (Paraform. 1)    CD62        12         16    GP53        18         22    PDGF        20         24    1.8% Paraformaldehyde/60 minutes (Paraform. 2)    CD62        6, 3       12, 7    GP53        14, 19     21, 30    PDGF        12, 18     18, 40    2.0% Paraformaldehyde/120 minutes (Brinkhous)    CD62        11          6    GP53         1          1    PDGF        27         30    ______________________________________

From Table 3, it can be seen that the concentration and time ofstabilization results in platelets with substantially differentproperties. Paraform. 1 and paraform. 2 platelets showed an increasingnumber of activation markers present on platelets in blood circulatedacross the subendothelial surface of the vessel wall. Paraform. 2 showsa doubling of the activation markers following exposure to an activatingsurface. In contrast, the Brinkhous preparation shows essentially noactivation post circulation with the CD62 marker and minimal change inPDGF.

EXAMPLE 5 (COMPARATIVE EXAMPLE B) Use of Activation Markers toCharacterize Platelets Prepared with Potassium Permanganate

The purpose of this Example is to demonstrate that platelets fixed withpermanganate in accordance with the present invention release theirgranular contents after contacting a thrombogenic surface, while priorart platelets do not. This example was carried out in essentially thesame manner as Example 4 above, except with platelets fixed withpermanganate as described in Example 3 above. Three differentpermanganate-fixed platelet preparations were employed: plateletsstabilized with 0.02M permanganate and lyophilized in the presence ofTrehalose (Perm. 1); platelets stabilized with 0.02M permanganate andlyophilized in the presence of human serum albumin (Perm. 2); andplatelets stabilized with 0.01M permanganate and lyophilized in thepresence of human serum albumin (Perm. 3). For comparative purposes,platelets were also fixed with 2% paraformaldehyde for 120 minutes anddried in the presence of bovine serum albumin. Data are given in Table 4below.

From Table 4 it can be seen that, in contrast to permanganate-f ixedplatelets of the present invention, the Brinkhous platelet preparationshowed essentially no activation following exposure to a thrombogenicvessel.

                  TABLE 4    ______________________________________    Comparison of Permanganate Stabilized Platelets    with Activation Markers.              CD62 Marker (% Positive Platelets)    Platelets   Pre-Circulation                            Post-Circulation    ______________________________________    Perm. 1     39          86    Perm. 2     31          60    Perm. 3     ND          37    Brinkhous   11          6    ______________________________________

EXAMPLE 6 (COMPARATIVE EXAMPLE C) Adherence of Rehydrated Platelets toVessel Subendothelium

The ability of fresh and rehydrated platelets to adhere to exposedsubendothelial vessel wall was tested in an annular perfusion chamber.the Brinkhous platelet preparation and platelets prepared as describedin Examples 4 and 5 above, were compared. Platelets were removed fromACD anticoagulated whole blood and replaced with various preparations ofdried and rehydrated platelets. Whole blood and blood containingrehydrated platelets was then pumped through chambers containing severaleverted vessels. The flow and shear rates were constant for allpreparations. Results are shown in Table 5 below.

                  TABLE 5    ______________________________________    Adherence of Rehydrated Platelets to    Vessel Subendothelium.    Platelet Type Percent Coverage    ______________________________________    Fresh         53-76    Perm. 2       26-53    Paraform. 2   23-43    Brinkhous     44-80    ______________________________________

Although variability was high between runs, it is evident that theBrinkhous platelets were "sticker" than the other preparations. Allpreparations adhere to vessel subendothelium but less area of theexposed vessel wall is covered by platelets prepared by the presentinvention. Of course, adherence of the prior art platelets, which aremetabolically "dead," is a passive property which would not be followedby an appropriate metabolic response.

EXAMPLE 7 (COMPARATIVE EXAMPLE D) Hypotonic Shock Recovery Test

The hypotonic shock recovery test is used to assess the ability ofplatelets to remove water and recover from swelling caused by increasedwater uptake by the platelet. To measure the extent of recovery fromswelling, platelet suspensions at 3×10⁸ /mL in citrated plasma weretreated with 1/2 volume deionized water in a Chronolog aggregometer at37° C. The light transmittance signal (%T) increases immnediately withthe uptake of water by platelets due to hypotonic shock (%T_(max)),followed by a return of %T to near baseline (%T_(base), corrected fordilution) as the water is actively expelled by intact platelets. Theextent of recovery after 10 minutes was quantified as: ##EQU1## The rateof recovery from hypotonic shock was a separate measurement, carried outas above except at 22° C. in an unstirred Payton aggregometer. The rateof recovery was computed as the rate of change in %T from 1 minute to 3minutes after addition of deionized water. The results given in Table 6are expressed as a percentage of the rate of change in %T obtained withfresh platelet controls. The rate and extent of recovery of platelets inhypotonic shock tests depends largely on membrane integrity and residualmetabolic activity. The Brinkhous platelet preparations werenon-responsive; while platelet preparations of the present inventionshowed 40 to 100% recovery.

                  TABLE 6    ______________________________________    Hypotonic Shock Recovery of Different    Stabilized Platelet Preparations.    Platelet         Recovery    Preparation      Rate    Extent    ______________________________________    Perm. 1           0-10   40    Perm. 2          0-8     --    Paraform. 2      26-43   100    Brinkhous        0        0    ______________________________________

EXAMPLE 8 (COMPARATIVE EXAMPLE .E) Thrombin Generation by PlateletPreparations

The purpose of this Example is to illustrate that small changes inconcentration of fixative and time of fixation result in a differentresponse of platelets to stimuli.

Four platelet preparations were tested for thrombin generation (which isplatelet concentration dependent) and support of the prothrombinasecomplex on the surface of the platelets. Results are shown in FIG. 1.Platelet count ×1000 is given on the horizontal axis; thrombingeneration in units is given on the vertical axis. The preparationswere: (i) paraform. 1 platelets (triangles in FIG. 1); (ii) paraform. 2platelets (squares); (iii) Brinkhous platelets (diamonds); and (iv)fresh, washed platelets as a control (circles). Paraform. 2 plateletsshowed the maximum rate of thrombin generation followed by paraform. 1platelets, and then Brinkhous platelets. Control platelets showed thelowest rate of thrombin generation.

EXAMPLE 9 Expression of Platelet-Derived Growth Factor (PDGF) on Surfaceof Fixed-Dried Platelets

The purpose of this Example was to examine platelets fixed and dried byvarious means for the expression of PDGF on the surface thereof.Antibodies and platelet preparations are as described in Examples 5 and6 above; data is given in Table 7 below.

                  TABLE 7    ______________________________________    Expression of PDGF on Platelet Surface.              Percent Platelets Positive for PDGF    Preparation Pre-Circulation                            Post-Circulation    ______________________________________    Fresh Blood 45          67    Perm. 2     ND          64    Paraform. 1 37          34    Paraform. 2 28          40    Brinkhous   27          30    ______________________________________

These data indicate that both the Brinkhous platelets and platelets ofthe present invention express PDGF on the surface thereof, and that thePerm. 2 and Paraform. 2 platelets express more PDGF on the surface thanBrinkhous platelets before or after stimulation.

EXAMPLE 10 Topical Administration of Fixed-Dried Platelets Which ExpressPDGF to Facilitate Wound Healing in Pig

This example describes the use of platelets which express PDGF on thesurface thereof to facilitate wound healing in pigs.

Porcine platelets used in this experiment were prepared essentially inthe manner described in Example 1 above, modified as set forth below. Toremove the paraformaldehyde, an equal volume of imidazolebuffered-saline, (IBS) pH 7.35, is added. The platelets are pelleted bycentrifugation at 1500×g for 8 minutes at room temperature. Thesupernatant is discarded and the pellet resuspended in 5-10 ml IBS, pH6.8. The wash was repeated twice. The procedure is then the same asdescribed in Example 1, except the concentration of porcine platelets is8×10⁴ /μl. Porcine albumin was added as a stabilizer for drying, in anamount similar to that described in Example 2.

The platelet dressing was prepared using BIOBRANE II™ surgical dressingon 1 square centimeter sections (Don B. Howland, Inc. Sugarland, Tx.) in10 cm diameter petri dishes. 1 cm² of the dressing was saturated with3.2×10⁹ porcine platelets by pipetting 1 ml of solution containing thesaid amount of platelets on the dressing, then carefully transferringthe dressing with the platelets into a lyophilization chamber, and thematerial dried at -40° C. until a cracked white powder was evident.

Two adult pigs were studied. The pigs were anesthetised and controlledpunch wounds were made using a dermatologic 3 millimeter (mm) punch. Onday 0, wounds of 3 mm deep ×3mm wide were produced in the shavedsterilized area along the back of anesthetised pigs. The woundpenetrates the epidermis, dermis and fatty tissue. Three rows of 6wounds each were produced. Row 1 was treated with dried platelets. Row 2was treated with mesh impregnated with dried platelets. Row three wasleft untreated. On day 1, wound one of each row was removed. On day 2,wound 2 of each row was removed. On each day for the next 4 days eachsucceeding wound of each row was removed for study. The removed tissuewas fixed with formaldehyde and treated with standard histologictechniques, stained and examined microscopically for evidence of woundrepair. Each section was examined for presence of platelets,fibroblastic proliferation along the sides and at the bottom of thewound, epithelial regrowth and evidence of inflammatory response. Theamount of platelets packed into each site was not determined exactly.Wounds in rowone were filled with dried platelets. Each punch wound wasfilled to capacity with dried platelets. Wounds in row 2 were treated bypacking 1 cm² mesh containing dried platelets into each wound.

Qualitatively, all the treated sections showed advanced repair ascompared to the untreated wounds.

EXAMPLE 11 Intraveneous Administration of Fixed-Dried Platelets Normaland von Willebrand Disease Dogs

To measure in vivo hemostatic effectiveness of rehydrated platelets, twonormal dogs and one von Willebrand factor deficient (von Willebranddisease, vWD) dog were infused with rehydrated fixed-dried normal canineplatelets. The canine platelets were fixed, dried and reconstituted inessentially the same manner as human platelets as described in Examples1 and 2 above, except that the platelets were fixed in 0.67%paraformaldehyde solution for one hours. Table 8 below shows thephysical data relative to the infusion and characteristics of the dogs.

                  TABLE 8    ______________________________________    Infusion of Rehydrated Platelets (RP) into Normal (N) and    von Willebrand Disease (vWD) dogs.                     Phenotype    Physical Data      N        N      vWD    ______________________________________    Weight (Kg)        20       15.9   9.1    Platelet Count (×10.sup.6)                       255      225    285    Total Circulating Platelets (×10.sup.9)                       448      315    228    RP Infused (ml)    10       9.2    8.5    Total RP (×10.sup.9)                       82.5     138    117    Total RP (% Original Platelets)                       18.4     43     51    % vWF (initial)    100      100    0    % vWF (post cryo.) --       --     50    BT (min:sec) Initial                       2:28     1:55   >15    BT (min:sec) post cryo.                       --       --     7:52    BT (min:sec) post.cryo., post RP                       2:52     1:55   6:40    ______________________________________

Following infusion of platelets into dogs, above, blood samples werecollected throughout the experiment, approximately four hours, asfollows. An anesthetized dog is infused with rehydrated platelets. Thecarotid arteries are exposed. A cannula is placed in the femoral arteryfor measuring blood pressure and a second cannula is placed in thefemoral vein for sample collection and administration of fluids. Onecarotid artery is subjected to a pinch injury with added stenosis inaccordance with known techniques (See Nichols et al., CirculationResearch 59, 15-26 (1988)). The dogs are monitored for changes in bloodpressure, heart rate, respiration etc. to evaluate any adverse responseto infusions of fixed-dried platelets. Rehydrated platelets are labeledwith a fluorescent dye, infused and blood samples collected to examinefor presence of labeled rehydrated platelets in the peripheralcirculation, in the formed thrombus, in areas of the disrupted vesselwall (subendothelium) and adherent to normal vessel wall. Cuts were madein the margin of the dog's ears and sections were prepared formicroscopic examination for adherent rehydrated platelets. Table 9 showsthe results of those studies.

                  TABLE 9    ______________________________________    Effect of Infusion of Rehydrated Platelets (RP) into Normal    (N) and von Willebrand Disease (vWD) dogs.                     Phenotype    Test               N        N      vWD    ______________________________________    Transfusion Reaction                       none     none   none    RP In Peripheral Circulation                       yes      yes    yes    RP Present in Formed Thrombi                       yes      yes    yes    RP Adhesion to Subendothelium                       yes      yes    yes    RP Adhesion to normal vessel wall                       no       no     no    RP Adhesion to cut surface                       yes      yes    yes    ______________________________________

The foregoing examples are illustrative of the present invention, andare not to be construed as limiting thereof. The invention is defined bythe following claims, with equivalents of the claims to be includedtherein.

That which is claimed is:
 1. A surgical aid, comprising, in combination:a solid, physiologically acceptable substrate; and fixed, dried blood platelets carried by said substrate, wherein said platelets express platelet-derived growth factor on the surface thereof.
 2. A surgical aid according to claim 1, wherein said substrate is selected from the group consisting of wound dressings, sutures, fabrics, and prosthetic devices.
 3. A surgical aid according to claim 1, wherein said surgical aid is sterile, and wherein said surgical aid is packaged in a sterile container. 